【作者】 韩俊；

【导师】 王明丽； 龚蕴贞； 史百芬；

【作者基本信息】 安徽医科大学 ， 病原生物学， 2001， 硕士

【摘要】 目的：构建人巨细胞病毒（HCMV）AD169株及地方株基因组cDNA表达文库，为进一步研究其基因组的结构特点及功能创立基本条件。方法：从MOI=10人巨细胞病毒（HCMV）感染HF细胞96小时，提取mRNA OD260/OD280比值为2.1，逆转录合成cDNA片段，重组入λgtll EcoR酶切位点之间，包装蛋白包装，转染宿主菌LE392和Y1090；经IPTG诱导和x-gal颜色选择测定。采用鼠多克隆抗血清进行免疫学筛选和地高辛标记特异性寡核苷酸探针进行原位噬斑杂交筛选阳性克隆，经PCR扩增鉴定。结果：构建的cDNA表达文库的库容量分别为3.6×10~6pfu/ml和3.3×10~6pfu/ml，经检测HCMV AD169 cDNA文库的重组效率为76%；免疫学筛选出阳性克隆为168；pp65核酸探针进行噬斑杂交筛选出34个阳性克隆；HCMV pp65全长PCR扩增出3个阳性克隆。结论：通过抗血清和核酸杂交筛选证实成功的构建了HCMV AD169株感染96小时cDNA表达文库，并从AD169文库中筛选出3个pp65阳性克隆。本研究结果将为阐明HCMV在机体内的免疫逃逸机制；病毒基因及产物与细胞相互作用；探索新型疫苗奠定基础。更多还原

【Abstract】 Objective: Construct HCMV AD 169 strain and isolated strain genome cDNA expressing library, which provide a sound basis for further studying of the structural character and function of their genome Methods: HF cell which were infected by 1-ICMV AD169 sftain(MON1O) and HCMV isolated strain(MOI=1O)cells were collected at 96h p.i, mRNA contained poly(A) was isolated by chronimatography on oligo(dT) cellulose, whose ratio of 0D260/0D280 was 2.1, then reverse transcripted into eDNA, second- strand DNA synthesis was done by using Rnase H and DNA polymerase 1, and E. co/i ligase to replace the RNA strand with deoxynucleotides . After methylation and EcoR I linker addition the cDNA was cloned into EcoR I-digested lambda gtl 1 .The ligated DNA was packaged , then transinfected host cells ,ie LE392 and Yl 090.The libray was screened by IPTG inducing and X-gal colouring , immune blot with anti-HCMV mouse convalescent sera , nucleic acid hybridization with DIG- labled HCMV pp65 gene probe and confirmed by PCR. Results: HCMV eDNA expressing libraries were constructed , their volume were 3.6 X lO6pfulml and 3.3 X 1 O6pfuIml respectively,recombinatant efficiency of AD 169 was up to 76%, 168 positive clones of ADI 69 were tested by immune blot, 34 positive clones were obtained by dot nucleic acid hybridization with pp65 probe, 3 positive clones were amplified by HCMV pp65 all length primer. Conclusion:HCMV AD169 strain and isolated strain cDNA expressing library have been constructed and 3 pp65 positive clones frome AD 169 cDNA expressing library had been screened.These results should be valuable for studying HCMV immunology mechanism, effect of viral genes on cells and new vaccines.更多还原